【作者】 胡娇；

【导师】 焦新安；

【作者基本信息】 扬州大学 ， 预防兽医学， 2010， 硕士

【摘要】 从上世纪70年代遗传工程技术兴起以来,出现了许多新的疫苗研究策略。减毒沙门菌作为疫苗载体具有安全、高效的特性,已受到医学与兽医学界的广泛重视。减毒沙门菌的主要优点包括它可以经黏膜途径免疫(口服或鼻内),操作方便,对接种宿主刺激小。此外,沙门菌为兼性胞内寄生细菌,能有效呈递抗原,激发机体抗沙门菌和外源蛋白的特异性体液与细胞免疫反应,并能同时诱导黏膜免疫与全身免疫。为避免在基因工程活菌苗的研制中使用抗生素作为选择压,将减毒沙门菌用于宿主载体平衡致死系统中,使在没有抗生素存在的情况下,重组质粒能稳定存在于宿主体内。这样的疫苗设计为防控重大的动物疫病带来了新的希望。猪霍乱沙门菌减毒株C500 (Salmonella choleraesuis C500 strain)是通过化学致弱用于预防仔猪副伤寒的有效活菌苗,其免疫原性强,安全性好,使用40余年来使我国仔猪副伤寒得到了较好的控制。因此,利用减毒的猪霍乱沙门菌C500构建宿主载体平衡致死系统用于运送重要疾病病原基因疫苗很有实际应用价值。新城疫(Newcastle disease, ND)是由副粘病毒科禽腮腺炎病毒属中的新城疫病毒(Newcastle disease viruses, NDV)引起多种禽类发生感染和高度死亡的一种急性、接触性传染病,常给养禽业造成重大经济损失。在许多国家对该病的防控主要靠疫苗的免疫接种。NDV的融合蛋白F介导病毒与宿主细胞膜的融合,是病毒入侵细胞的重要分子。本研究构建了以猪霍乱沙门菌C500△asd缺失株为运送载体的NDV F基因工程菌,以小鼠和鸡为动物模型,评价了其免疫特性,初步探索了猪霍乱沙门菌C500△asd缺失株做为活疫苗载体的可行性。1猪霍乱沙门菌C500△asd缺失株宿主载体平衡致死系统的构建与鉴定根据GenBank中已发表的猪霍乱沙门菌asd基因的序列,设计两对引物分别扩增asd基因的上下游片段,然后构建含缺失1,107 bp的asd基因与蔗糖敏感基因(sacB)的重组自杀性质粒,与猪霍乱沙门菌C500接合转移,两步法筛选无抗性的△asd缺失株,用PCR证实基因组asd基因的缺失突变。该缺失株生长必需外源DAP(二氨基庚二酸)。进一步鉴定猪霍乱沙门菌C500△asd缺失株的表型、生长特性、毒力等,结果表明猪霍乱沙门菌C500△asd缺失株与其亲本株猪霍乱沙门菌C500的表型、生长特性基本一致,而其毒力有了进一步的下降。将携带asd基因的红色荧光蛋白质粒转化猪霍乱沙门菌C500△asd缺失株后,在体外传至100代后仍可高效表达红色荧光蛋白,说明该缺失株可以用来作为宿主载体平衡致死系统来高效表达外源基因,为深入研究以猪霍乱沙门菌C500△asd缺失株作为疫苗载体奠定了基础。2新城疫病毒F蛋白在猪霍乱沙门菌C500△add缺失株中的表达及部分免疫特性分析复苏本实验室保存的重组菌X4550(pYA3334-F),进行PCR和SDS-PAGE鉴定,阳性菌提取重组质粒(pYA3334-F)电转化猪霍乱沙门菌C500△asd缺失株,普通LB平板筛选阳性克隆,命名为猪霍乱沙门菌C500△asd(pYA3334-F).SDS-PAGE电泳检测到F蛋白的表达。动物免疫实验结果表明,重组菌猪霍乱沙门菌C500△asd(pYA3334-F能在小鼠和鸡体内诱导产生针对F蛋白的特异性抗体,显示本研究构建的以猪霍乱沙门菌C500△asd缺失株运送的外源抗原具有良好的免疫原性,能够激发有效的免疫应答,初步证明该系统做为活疫苗载体的可行性。更多还原

【Abstract】 Since 1970s, many new vaccine strategies were developed with the advent of genetic engineering, so the safer and more efficient live vaccine using attenuated Salmonella spp. as the vector received wide attention in medical and veterinary medicine. The major advantage of the attenuated Salmonella spp. is that it can be administered by mucosal route (oral or intranasal), easily genetically manipulated and cause little side effect on animals. In addition, as an intracellular invasive bacterium, Salmonella spp. can effectively present antigen, induce specific humoral and cellular immune response against Salmonella spp. and heterologous antigen, and trigger both mucosal and systemic immune at the same time. To avoid using antibiotic as the screening marker in the construction of genetic engineering live vaccine, nutrition vicious attenuated Salmonella spp. based on balanced-lethal host-vector system was designed to deliver the expressed protein of foreign gene. Such vaccine design brings new hope to animal under the threat of contagious disease.Strain C500 of Salmonella choleraesuis is an avirulent vaccine strain attenuated by chemical method, it has good immunogenicity and safety and has been used to prevent piglet paratyphoid in China for over 40 years. Strain Salmonella choleraesuis C500 used as a delivery system for heterologous antigens based on the host-vector balanced lethal system would have great prospect in the near future. Newcastle disease (ND) is a highly contagious disease that causes substantial economic losses for poultry. Newcastle disease virus (NDV) belongs to the newly defined genus Avulavirus in the family of Paramyxoviridae. Its vaccine is frequently used for the prevention and control of ND in some countries when the disease spreads widely. The F protein, which mediates fusion of viral envelope and cellular membrane, is an important factor for viruse invasion and also a good protective immunogen. In this work, Salmonella choleraesuis C500△asd mutant was constructed, and used as a delivery system for F gene based on the balanced-lethal host-vector system. The immune responses of i.n. and i.m. vaccination with the recombinant Salmonella choleraesuis C500 vaccine strain expressing F antigen of NDV both in mouse and chicken were invaluated, the results showed that both of them produced specific antibody against F protein. It suggests that this balanced-lethal host-vector system of Salmonella choleraesuis could express foreign gene(s), delivery the target protein and intiate specific immunity, and could be used as vaccine vector against infectious disease.1 Construction and characterization of Salmonella choleraesuis C500△asd host-vector balanced lethal systemTwo sets of primers were designed and synthesized based on the sequences of asd gene of Salmonella choleraesuis in GenBank, then the upstream and downstream fragments of asd gene were amplified and cloned from Salmonella choleraesuis C500 genome DNA. The recombinant suicide plasmid was constructed in which asd gene was replaced by Cm gene. Strains with the deletion of asd gene were selected by two-step protocol and named Salmonella choleraesuis C500△asd mutant, which could not live without additive DAP. The mutant was confirmed by PCR method. Its phenotypes, growth properties and virulence characteristics were further verified, the results showed that the chracteristics of△asd mutant were consistent with those of its wild type, and its virulence had a drop compared with its parent strain. The dsRED plasmid was transformed into Salmonella choleraesuis C500△asd mutant by electroporation to generate Salmonella choleraesuis C500△asd (pYA3334-dsRed), the recombinant could express dsRED stably and efficiently after 100 generations in vitro. The results showed that the Salmonella choleraesuis C500△asd mutant was successfully constructed and it could be used as a vector based on the host-vector balanced-lethal system to express foreign gene(s) stably and efficiently, this study would pave a way for developing Salmonella choleraesuis C500 vaccine strain as a live vectored vaccine.2 Expression of F protein of Newcastle disease virus in Salmonella choleraesuis C500△asd mutant and analysis of its immunogenicityThe recombinant Salmonella typhimurium X4550(pYA3334-F) was indentified by PCR and SDS-PAGE, the positive plasmid pYA3334-F was extracted and electroporated into Salmonella choleraesuis C500△asd mutant, the recombinant was screened and designated as Salmonella choleraesuis C500△asd (pYA3334-F), SDS-PAGE was conducted to detect the expression of F gene in recombinant Salmonella choleraesuis C500△asd (pYA3334-F). It was shown that Salmonella choleraesuis C500Aasd (pYA3334-F) induced specific antibody against F protein of NDV both in mouse and chicken.In summary, the F protein expressed in attenuated Salmonella choleraesuis C500△asd had good immunogenity, and could initiate humoral and celluar immune responses. Salmonella choleraesuis C500△asd might be used as a live vaccine vector.更多还原